The in vivo conversion of the cellular prion protein (PrPC) into an alternate conformer, the infectious prion (PrPSc), is the etiologic event in prion replication and pathology. Until recently, in vitro conversion protocols have failed to yield infectious prions from recombinant sources of wild-type PrP or segmental deletion variants of PrP. Recently, we have shown that in vitro converted, recombinant wild-type mouse PrP(89-230) can cause a historically accurate neurodegenerative disease that is serially transmissible. This observation creates several opportunities for advancing biophysical studies of prions. In vitro prions show unusual strain characteristics, and by inference structural properties, that are maintained when passaged in wild-type or transgenic mice. We propose to study the structure of these synthetic prions by fiber diffraction, electron crystallography, and molecular modeling. Currently, the in vitro conversion is still fairly inefficient. For this reason, we will focus our initial investigations on synthetic prions that have been passaged in mice. Once the efficiency of the in vitro conversion process is improved, we plan to carry out additional experiments to characterize synthetic prions directly from recombinant sources. Our initial experiments are designed to analyze synthetic prions that form amyloid fibers and two-dimensional (2D) crystals and to compare these results with those for naturally occurring prions. The experimental data will be used to refine structural models of PrPSc that are based on structures of known proteins or domains of proteins. In particular, we will focus on the left-handed parallel beta-helix as a motif that can account for the secondary structure constraints implied by optical spectroscopy data and spatial constraints determined by electron crystallography. Historically, fiber diffraction results have contributed only low-resolution data to our analysis of the structure of PrPSc. However, we believe that when coupled with modeling and electron crystallography data, we will be able to extract more meaningful information from these studies. Therefore, we plan to revisit fiber diffraction studies using more advanced methods of fiber alignment coupled with synchrotron-based X-ray and electron diffraction approaches.